Natural resveratrol analogs differentially target endometriotic cells into apoptosis pathways

The specific characteristics of endometriotic cells are their ability to evade the apoptotic machinery and abnormal response to apoptotic stimuli. Natural-originated compounds may constitute a beneficial strategy in apoptosis modulation in endometriosis. We investigated and compared the potency of natural resveratrol analogs, including piceatannol, polydatin, and pterostilbene, in targeting cell death pathways, including apoptosis-related morphologic and biochemical processes, alongside the modulation of the critical genes expression. Upon resveratrol and pterostilbene treatment, a significant reduction of endometriotic cell viability and an increased apoptotic proportion of cells were noted. The lower antiproliferative potential was found for piceatannol and polydatin. Endometrial stromal T HESC cells were significantly more resistant than endometriotic epithelial 12Z cells to the cytotoxic activity of all analyzed compounds. They differentially affected endometriotic cell viability, cell cycle, anti- and proapoptotic genes regulation, caspases expression and enzymatic activity, and DNA fragmentation. Pterostilbene-mediated endometriotic cell apoptosis modulation was confirmed to be most effective but without evident caspase 3 upregulation. Our study provides valuable insight into the apoptogenic activity of resveratrol and its natural analogs in endometriotic cells. Data obtained revealed the highest therapeutic potential of pterostilbene by effectively targeting cell death determinants in endometriosis, strengthening its optimization in further extensive research.


Results
Effect of resveratrol and its derivatives on endometriotic and endometrial cell viability. The effect of resveratrol and its natural analogs on the viability of endometriotic epithelial 12Z cells and endometrial stromal T HESC cells was assessed using the MTT assay. Table 1 presents the IC 10 and IC 50 values (the inhibitor concentration required for 10% and 50% reduction in enzyme activity) obtained after 48-h incubation with the tested compounds. In 12Z cell culture, the IC 10 and IC 50 values of piceatannol and polydatin were significantly higher than those of resveratrol. The cytotoxicity of resveratrol and pterostilbene to 12Z cells was not markedly different. T HESC and 12Z cells varied in susceptibility to the treatment with stilbenes tested, as evidenced by the cytotoxic doses shown in Table 1. Pterostilbene revealed the highest cytotoxic potential for T HESC cells, significantly higher than the parent compound. However, comparing IC 50 values, pterostilbene cytotoxicity in T HESC cell culture was almost twofold lower than in 12Z cell culture. Similarly, resveratrol evoked much weaker cytotoxic effects in T HESC cells than in 12Z cells. Polydatin at concentrations up to 400 µM was not cytotoxic Table 1. Effect of resveratrol, pterostilbene, piceatannol, and polydatin on endometriotic epithelial (12Z) and endometrial stromal (T HESC) cell viability, determined by the MTT reduction assay. *p < 0.05, **p < 0.001 significance of differences between cell treatment with resveratrol analogs compared to resveratrol. # p < 0.05, ## p < 0.001 significance of differences between cytotoxic doses of stilbenes to 12Z and T HESC cells.

Tested compounds Endometriotic cells-12Z
Endometrial cells-T HESC IC 10  www.nature.com/scientificreports/ to the T HESC cells, while at a dose of 86.76 µM, it induced the first cytotoxic symptoms in 12Z cells. It is worth noting the Alamar Blue assay proved a similar cytotoxic effect of analyzed compounds in 12Z and T HESC cells, although it was less sensitive than the MTT assay. The increased susceptibility of endometriotic 12Z cells than endometrial T HESC cells to resveratrol and its natural analogs was consistent in both assays (Fig. 1). To summarize, resveratrol, pterostilbene, and piceatannol had a more apparent toxic effect on endometriotic cells than endometrial cells.

Effects of resveratrol and its analogs on the endometriotic cell monolayer morphology.
After the treatment of resveratrol and its analogs, morphological changes in the endometriotic cell monolayer were observed under the inverted phase contrast microscope (Fig. 2). As the concentration of the analyzed compound increased, the cell culture became sparsely populated and defective. The most significant disturbances in the development of the endometriotic cell monolayer were detected following treatment with resveratrol and pterostilbene. Inhibition of adherent cell growth, losing contact with adjacent cells, and typical apoptotic features, such as rounding and membrane blebbing, were recorded dependently on the dose of stilbene tested. The arrested proliferation and echinoid spikes on the apoptotic cell surface and rampant death of endometriotic cells were observed, especially after exposition to high resveratrol and pterostilbene dosage. However, the first disturbances in 12Z cell monolayer development were noted after cell treatment with cytotoxic doses as low as 10 µM (Fig. 2).

Microscopic detection of endometriotic cell apoptosis.
The apoptogenic properties of the tested compounds were monitored using fluorescence microscopy analysis of treated endometriotic 12Z cells after staining with DAPI (Fig. 3). The cells displayed common characteristics typical for apoptosis, such as chromatin condensation and cleavage, formation of pyknotic bodies of condensed chromatin, and cell disintegration into apoptotic bodies and microvesicles. The results indicated evident changes typical for apoptotic cells, such as increased nuclear condensation, formation of apoptotic bodies, and fragmentation in multiple segregated body features.

Effect of resveratrol and natural analogs on endometriotic cell death induction.
After treating endometriotic 12Z cells with stilbenes, the phosphatidylserine externalization was measured by Annexin V and PI double staining (Fig. 4A). Examples of the single-cell images from sub-populations: live cells (Annexin V/   Fig. 4C show that resveratrol and its derivatives dose-dependently decreased the population of live cells and simultaneously increased the population of early and late apoptotic cells with the limited population of mechanical necrotic cells. Under treatment with resveratrol, pterostilbene, and piceatannol at concentrations of 50 µM, 100 µM, and 200 µM, a significant dose-dependent increase in the population of late apoptotic cells was observed in treated 12Z cell cultures. Pterostilbene was the most efficient apoptosis inducer based on the overall apoptotic rate parameter. In the 12Z cell culture exposed to pterostilbene at a dose of 200 µM for 48 h, approximately 94% of treated cells were classified as apoptotic cells. In contrast, 12Z cells treated with polydatin underwent apoptosis with an increased population of early apoptosis cells without induction of late apoptosis (Fig. 4C).

Effect of resveratrol and natural analogs on DNA fragmentation. Cell death via apoptosis in 12Z
cells after exposure to resveratrol and its natural derivatives was determined by detecting DNA fragmentation (Fig. 5). DNA degradation was correlated in a dose-dependent manner. TUNEL assay showed increased DNA fragmentation in endometriotic 12Z cells treated for 48 h with resveratrol, pterostilbene, and piceatannol. The most significant DNA strand breaks were observed in the cells exposed to resveratrol and piceatannol at the concentration of 100 µM (42.8% and 49.6%, respectively) and 200 µM pterostilbene (62.6%). In contrast, polydatin did not induce DNA damage in treated 12Z cells (1.08%). Tunel-positive cell population in the 12Z cell culture treated with camptothecin was estimated at 32.5%, compared to 0.6% in the vehicle-treated control culture.

Effect of resveratrol and its analogs on cell cycle progression.
To investigate whether resveratrol and its analogs have interfered with cell cycle progression in endometriotic 12Z cells, we analyzed the cell cycle distribution by flow cytometry. The obtained data showed that treatment with resveratrol and its analogs caused a significant increase in an apoptotic cell population with lower DNA content and simultaneously a decrease in cell population in G 1 /G 0 cycle phase depending on the compound concentration, except for polydatin, which did not increase the number of apoptotic cells in a dose-dependent manner (Fig. 6). The largest subpopulation of apoptotic cells was found in the cell cultures treated with resveratrol and pterostilbene at the concentration of 100 µM (31.2 ± 8.0% and 33.1 ± 9.7%, respectively) and piceatannol at a concentration of 200 µM (45.1 ± 5. 6%).

Effect of resveratrol and its analogs on the expression of genes involved in apoptosis.
The effect of resveratrol and its natural analogs on the expression of pro-or antiapoptotic regulators, was examined by real-time PCR. The analysis of mRNA expression showed that the analyzed compounds affect the expression of several proapoptotic genes and genes of two pro-survival proteins. The enhanced expression of pro-survival B-cell lymphoma 2 (BCL-2) and BCL2-like 1 (BCL2L1) genes was observed in 12Z cells treated with resveratrol (50 µM and 100 µM), pterostilbene (50 µM and 100 µM), and piceatannol (50 µM, 100 µM, and 200 µM) (Fig. 7). On the other hand, these stilbenes significantly increased the mRNA expression of the BAX gene, which promotes apoptosis. The highest BAX mRNA expression was observed in 12Z cells treated with pterostilbene at     www.nature.com/scientificreports/ Moreover, after 12Z cell treatments with resveratrol and pterostilbene (50 µM and 100 µM) and piceatannol (200 µM), the mRNA expression of death receptor FAS was significantly increased. The FAS overexpression depended on the concentration of stilbenes, reaching a relative expression up to tenfold higher in treated endometriotic cells. The role of proteases in resveratrol-, pterostilbene-, and piceatannol-induced apoptosis was also indicated by the upregulation of well-known initiator caspases of the mitochondrial and death receptor apoptotic pathways such as caspases 8, 9 and executioner caspase 3 (Fig. 7). Polydatin did not significantly increase mRNA transcripts of the analyzed BAX, FAS, CASP3, CASP8, and CASP9 genes.

Effect of resveratrol and its analogs on the activity of caspases-3/7. The effect of resveratrol and its
analogs on the effector caspases-3/7 activity in endometriotic cells is shown in Fig. 8. Resveratrol evoked a dosedependent caspase-3/7 activation in 12Z cells (F = 2467.29, p < 0.0001). The most potent up-regulating effect was observed under treatment with resveratrol at a concentration of 100 µM, which caused a 10.3-fold increase in caspase-3/7 activity. It was consistent with the results of CASP3 gene expression activation. As shown in Fig. 8, pterostilbene at a concentration of 100 μM modestly activated caspases 3/7, although it inhibited their activity at 200 μM. There was a 2.8-fold activation of caspase 3/7 enzymes following treatment with piceatannol at a 200 μM dose, which complies with the CASP3 mRNA expression level. Caspase 3/7 activity was not significantly changed in the 12Z cells by their exposure to polydatin.

Discussion
An important clue for developing new therapeutic strategies in endometriosis is that the eutopic and ectopic endometrium of endometriotic patients shows less susceptibility to apoptosis than the endometrium of nonendometriotic patients 18 . For this reason, manipulating programmed cell death processes by enhancing apoptotic stimulants of endometriotic cells could be a promising approach to treating endometriosis. This study was designed to evaluate the effects of natural analogs of resveratrol on the inhibition of endometriotic cell survival pathways and upregulation of cell death pathways, including apoptosis signaling.
The present study determined the cytotoxic effect of the analyzed stilbenes on human endometrial stromal cells (T HESC) and human endometriotic epithelial cells (12Z). The 12Z cell line retains characteristics of the active phase of endometriosis and is considered suitable for studying the cellular and molecular context of endometriosis 19 . In contrast, the T HESC line represents a distinct stromal compartment of the endometrium and resembles typical endometrial features; therefore, it has been widely applied in endometriosis studies 20,21 . However, endometriotic stromal cells were not employed in the experiments due to the lack of an established immortalized stromal cell line. Recently, the 22B cell line derived from the stromal compartment of endometriotic tissue was no longer available for current research because of the cessation of cell growth during culture 22 . A more recent study by Madanes et al. described the effect of resveratrol on human endometrial stromal St-T1b and epithelial 12Z cells 23 . Resveratrol at a concentration of 100 µM after 48-h treatment was found to reduce cell viability by 45.4% and 49.2% in 12Z and St-T1b cell cultures, respectively. The effects of resveratrol were analyzed on primary cultures of human endometrial epithelial cells obtained from eutopic endometrial biopsies of patients with endometriosis. After exposure to 50 µM and 100 µM resveratrol, cells from control women and endometriosis patients showed a significantly lower proliferative potential 24 . In other studies, resveratrol at concentrations up to 100 μM did not influence the viability of endometrial stromal cells derived from patients with endometriosis after 48-h treatment. Increasing the dose of resveratrol to 200 μM reduced endometriotic stromal cell viability to approximately 50% 25,26 . Resveratrol by itself did not induce apoptosis in the ectopic www.nature.com/scientificreports/ stromal cells; however, it enhanced the effect of TNF-α-related-apoptosis-inducing ligand (TRAIL) and acted by silencing survivin expression 26 . The control non-endometriotic T HESC cells derived from human endometrium were also applied in our cytotoxicity experiments since the distinction in the cellular response between endometrial and endometriotic tissue is an important issue in evaluating the activity of the therapeutic agent. Endometrial T HESC cells were more resistant to their cytotoxic activity than endometriotic 12Z cells. Based on the IC 50 values, the cytotoxicity of resveratrol was fivefold higher for the endometriotic 12Z cells than for the endometrial T HESC cells. Resveratrol treatment seems to have a different effect on endometrial and endometriotic cells, with a harmful impact directed to endometriotic cells. Nonetheless, diverse cell population compartment representativeness must be accentuated in assessing the susceptibility of endometriotic and endometrial cells.
The results presented in this work indicated that natural resveratrol analogs are cytotoxic to cells derived from the endometrium and endometriotic lesions with different degrees of potency. Pterostilbene, a dimethyl ether analog of resveratrol with better intestinal absorption and elevated hepatic stability, has been reported to regulate pathogenic pathways associated with carcinogenesis, hematologic diseases, neurological, and age-related disorders 27 . Pterostilbene inhibited the growth of highly malignant melanoma cells at a bioavailable concentration by interfering with the molecular signaling during cell division. Moreover, pterostilbene demonstrated activity in lymphoma cell lines with a mutation of the Fas gene, resistant to apoptosis induced by resveratrol and piceatannol 28 . Compared to resveratrol, fewer hydroxyl groups result in less susceptibility to conjugation metabolism; therefore, a longer half-life is predicted for pterostilbene 29 . However, until now, pterostilbene has not been investigated as an anti-endometriotic agent. Its cytotoxic effect on endometriotic 12Z cells was lower than resveratrol. On the other hand, pterostilbene inhibited the proliferation of endometrial T HESC cells more potently than the parent compound.
Plant-derived piceatannol has been identified as an antileukemic compound and a potent inhibitor of the proliferation and migration of vascular endothelial cells. As a result, piceatannol can limit the development of tumors by inhibiting neovascularization. Polydatin, the glycosylated form of resveratrol, can increase the quantity of trans-resveratrol due to the hydrolysis by β-glucosidase secreted by bacteria in the colon. In recent years, several studies have suggested that polydatin possesses a higher capacity than resveratrol to alleviate oxidative stress and has a more potent anti-inflammatory effect 30 . It was hypothesized that the additional hydroxyl group enhances the piceatannol reactivity and makes it a more potent free radical scavenger than resveratrol 28 . However, interference in various cellular targets and proven antitumorigenic effects 14,31 , piceatannol and polydatin biological activity are not yet determined in endometriosis. The potentially bioactive nature of the resveratrol analogs strongly justifies the research undertaking on their preventive and therapeutic effects on endometriosis. However, comprehensive and extensive research is needed to clarify molecular and cellular regulations of endometriotic cell growth and expansion treated with the proposed new alternative compounds.
We observed that under treatment with resveratrol and its analogs, the percentage of positive cells for Annexin V, an early sign of apoptosis, was increased significantly. Still, the most efficient increase in the population of apoptotic cells was caused by pterostilbene. Cumulative evidence has pointed out that pterostilbene shows stronger antiproliferative and apoptotic effects than resveratrol in human cervical cancer cells 32,33 . The experiments with pterostilbene ascertain it acts as a robust agent capable of regulating the p53-dependent apoptotic pathway and targeting the Nrf-2 pathway by developing endoplasmic reticulum-mediated stress in cervical cancer cells 32,34 . The superior inhibitory effects of pterostilbene compared to resveratrol were associated with the induction of p53 and p21, which contribute to cell cycle arrest at S and G 2 /M phases and subsequent reduction of cyclin E1 and cyclin B1 33 .
Our findings indicate that the first signs of an increase in the incidence of apoptosis were observed at 50 µM concentration of all analyzed compounds, identified by oligonucleosomal DNA fragmentation and accumulation www.nature.com/scientificreports/ on the histogram as subG1 apoptosis fraction. The DNA strand breaks represent a hallmark of the later stages of apoptosis, which are mediated by several endonucleases. Caspase-activated DNase (CAD) is expressed as a heterodimer with its inhibitor ICAD, cleaved by caspase-3 leading to CAD activation, degrades the nuclear material 35 . We analyzed the activation of caspases at the molecular and cellular levels. Resveratrol upregulated caspase-3 mRNA expression and proteolytic activity by 7.3-and 10.3-fold, respectively. Caspase 3 expression in pterostilbene-induced apoptosis in endometriotic cells was much weaker, even though this compound generated the most significant number of DNA strand breaks. Data on the apoptotic activity of resveratrol and its natural analogs for human T lymphoma cells revealed that the pan-caspase-inhibitor Z-VAD-fmk could inhibit apoptosis induced by trans-resveratrol, piceatannol, and 3′-hydroxypterostilbene but not apoptosis induced by pterostilbene, suggesting that it could activate apoptosis through a caspase-independent mechanism 36 . It has now been recognized that in response to apoptotic stimuli, mitochondria can also release caspase-independent cell death effectors such as apoptosis-inducing factor (AIF) and endonuclease G. Following release and translocation to the nucleus, AIF becomes an active executioner of the cell death by the initiation of large-scale DNA fragmentation 37 . It is essential to realize that apoptosis evolved complex death pathways, which overlap with each other. The traditional pathway involving BCL family proteins and caspases may recruit an additional response mechanism, such as caspase-independent AIF release, to facilitate the completion of apoptosis 37 . Overexpression of BAX was shown to trigger the release of AIF in 293 T and HeLa cells, suggesting that it can participate in multiple death paradigms 38 . According to this statement, in our study, pterostilbene was found to enhance BAX expression significantly. Different proapoptotic potentials between resveratrol and pterostilbene presented in this work are probably due to the structure-activity relationships. It has been found that the 3,5-dimethoxy motif facilitated the apoptogenic activity of the compound, even in cells expressing the multidrug-resistant phenotype or the antiapoptotic oncogene 36 .
An interesting inhibitory effect of piceatannol on the endometriotic cells was observed in our study. The experimental data suggest that apoptosis induction by piceatannol could be related to the extensive fragmentation of nuclear DNA in the treated 12Z cells, despite a relatively high IC 50 value (223.76 µM) indicating lower cytotoxic potency than resveratrol and pterostilbene. The only difference between the resveratrol and piceatannol is the presence of an additional hydroxy group in one of the piceatannol's aromatic rings. Piceatannol was found to exhibit potent tyrosine kinase inhibitory activity and inhibit a variety of tyrosine kinases involved in cell proliferation and overexpressed in different cancer cells 39 . Data obtained show that piceatannol at 100 µM led to almost 50% internucleosomal DNA fragmentation in 12Z cells with a significant increase in caspase-8, -9, and -3 mRNA expression. Similar observations concerning a minor piceatannol cytotoxic effect were made in hepatoma HepG2 cells at piceatannol doses ranging from 0 to 200 µM after 24, 48, and 72 h of treatment. Likewise, a significant percentage of apoptotic cells was detected in HepG2 cells treated with piceatannol at 100 µM and 200 µM, which also confirmed the TUNEL assay 40 . Our findings indicate that resveratrol, pterostilbene, and piceatannol at higher concentrations up-regulated the expression of the antiapoptotic BCL2 gene, and pterostilbene evoked the most substantial effect. Protein BCL-2 resides in the outer mitochondrial wall and inhibits cytochrome c release 41 . Pterostilbene was previously found to increase the expression of antiapoptotic BCL-2 and activate MAPK/ERK and PI3K/AKT signal pathways in human neuronal SH-SY5Y cells in neuroprotective experiments. Pterostilbene, which exhibits estrogenic activity, mediated neuroprotective potential via mainly ER-α receptor, which activated the BCL-2 expression presumably through genomic estrogen signaling 42 . The distribution of hydroxyl groups in aromatic rings in the stilbenes makes them similar in chemical structure to endogenous and synthetic estrogens such as 17β-estradiol (E2) or diethylstilbestrol 43 . One early identified biological target of resveratrol was the estrogen receptor (ER), and the compound was classified as a phytoestrogen. After dimerization of ER receptors and translocation to the nucleus, they bind to estrogen response elements (ERE) within target genes. The transcription of genes by ER can also be mediated through the non-classical genomic pathway by its direct binding to DNA with different transcription factors (e.g., AP-1, SP-1, or NF-κB) without binding to ERE. Genes involved in proliferation and survival or apoptosis, such as cyclin D1 and BCL2, are regulated by ER binding to the SP-1 44 . Many estrogenic compounds display varying degrees of agonism/antagonism depending on the cell type and target gene, and they are referred to as selective estrogen receptor modulators (SERMs). Resveratrol was thought to affect breast cancer cells through ER-dependent signaling pathways, some studies demonstrated that resveratrol analogs decreased triple-negative breast cancer cell viability in a dose-dependent manner, indicating the existence of an alternative, ER-independent mechanism 45 . The well-characterized endometriotic epithelial 12Z cells used in our experiments are ER-positive (ER-α, ER-β) 46 . Endometrial stromal T HESC cells were also found to express ER and were applied in analyzing the regulation of steroid receptors upon treatment with their antagonists and induction of decidualization 47,48 . Affecting mediators down-regulating ER signaling constitutes an essential direction for therapeutic purposes because estradiol at strikingly high quantities is the master regulator of endometriotic tissue pathology 49 .
Polydatin represents a natural precursor of resveratrol, in which the hydroxyl is replaced and occupied by a glucopyranoside ring, enabling much better bioavailability than resveratrol, benefiting from the way entering cells via glucose carriers 50 . A number of studies have suggested that polydatin therapeutic effect originates from its anti-inflammatory 51 , antioxidant 52 , and anticancer 53 activities. It remains unclear whether polydatin, like resveratrol, has antioxidation and antitumor properties or constitutes a clinically advantageous approach. In our studies, the effect of polydatin on endometriotic cell apoptosis was relatively minor. All analyzed compounds showed higher inhibition capacity in cell viability than polydatin did. Even though polydatin induced the early apoptosis stage under all measured concentrations (50 µM, 100 µM, 200 µM), cells did not go into the late apoptotic phase as under treatment with other stilbenes. Dose-independently, polydatin unaffected neither caspase activation nor expression of proapoptotic proteins. The reason that polydatin shows lower biological activity than resveratrol at the same concentration was investigated in the different tumor cells. The results www.nature.com/scientificreports/ indicated that polydatin was harder uptaken by the cells than resveratrol, and probably specific transporters in cells or lipid rafts endocytosis may determine its bioactivity 54 . Active compounds from natural products hold, in general, multiple molecular targets and can affect different signaling pathways involved in proliferation, apoptosis, invasion, migration, inflammation, reactive oxidative stress, and angiogenesis in the complex disease such as endometriosis 6 . New developed therapeutics for endometriosis will be based on the prevention of off-target toxicities like side effects on the eutopic endometrium or function of the reproductive tract. Therefore, the long-term safety of natural products, target pathway selectivity, and prevent recurrence will be assessed in future studies. Moreover, challenges should be addressed to improve bioavailability by implementing prodrugs, solid dispersions, and lipid-based formulation technologies 55 . Recently, some locally administered platforms were proposed to deliver drugs for endometriosis treatment, utilizing delivery technologies already explored in cancers. A polymeric drug delivery system to control the prolonged release of curcumin from nanofibers in the peritoneum and pelvic cavity was developed in a mouse model of endometriosis. Besides, in the rat endometriosis model, polymer/nucleic acid conjugates reduced the ectopic endometrial lesions in ectopic locations. The small-molecule drugs or siRNA can be targeted to the ectopic endometriotic tissue by the micelle systems and polymeric or peptide conjugates. Different nanoparticle structures (metal-oxide nanoparticles, silver nanoparticles) can be designed to recognize specific receptors in the lesions' microenvironment and target drug delivery 56 . Endometriosis drug delivery technologies are a relatively new approach but are essential for clinically translatable therapeutics for women's health applications.

Scientific
As discussed above, the study results indicate that resveratrol and its natural analogs may offer substantial preventive and therapeutic potential for treating endometriosis by specific induction of apoptosis via deathsignaling pathways. It is worth emphasizing that non-endometriotic T HESC cells were significantly less affected by resveratrol and its key phytoestrogenic analogs. The significant effects of pterostilbene on the proapoptotic events contributed to considerable inhibition of endometriotic cell proliferation, which was more preferential than resveratrol. The anti-endometriotic potency of pterostilbene has been unveiled and described for the first time in the presented work. Given the previous reports and our insights, we assumed more attention should be paid to the estrogenic activity caused by natural phytoestrogens in evaluating their bioactivity. We conclude that the properties of resveratrol and pterostilbene, involving the selective suppression of endometriotic cell proliferation and induction of apoptosis, support their preventive and therapeutic activity against endometriosis.

Materials and methods
Unless specified otherwise, all chemicals were purchased from Merck KGaA (Darmstadt, Germany). The cells were maintained routinely in phenol red-free DMEM/F12 supplemented with 10% charcoal/dextran treated FBS, 1% ITS + 1, and 50 mg/L gentamycin, at 37 °C and 5% CO 2 . Both cell lines were negative for mycoplasma contamination; mycoplasma-free cells were confirmed repeatedly during the study.
Cells were seeded at a density of 0.15 × 10 5 cells/cm 2 in 6-well plates. The 24-h cell cultures were treated with resveratrol and its derivatives for 48 h under standard culture conditions. Camptothecin was applied as a reference apoptosis inducer; its final concentrations were set to 5 µM and 50 µM.
Cell viability assay. Cell viability and metabolic activity were determined using the MTT colorimetric assay, according to the protocol described in previous studies 57 . Based on the obtained data, the dose-response curves were plotted, and inhibitory concentrations (IC) were calculated. In addition, cell proliferation was measured using the alamarBlue™ Cell Viability Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Fluorescence was measured using a Tecan M200 Infinite microplate reader (Tecan Group Ltd., Männedorf, Switzerland).

Microscopic detection of cell apoptosis. After treatment, cell monolayer morphology was assessed
under an inverted phase contrast microscope Axiovert 40C (Zeiss, Germany). Moreover, 12Z cells cultured on Millicell EZ SLIDE glass (Millipore, Merck KGaA) were subjected to the standard treatment protocol. Following exposure, cells were fixed with 4% paraformaldehyde, washed with PBS, permeabilized with 0.1% Triton X-100, and stained with 1 μg/ml DAPI. The cell nuclear morphology was assessed by fluorescence microscopic inspection (Axiovert 200, Zeiss, Germany).  Cell cycle analysis. Cell cycle progression was analyzed by flow cytometry using PI staining. After treatment, 12Z cells were collected, washed with PBS, and fixed in 70% ethanol. The cells were then washed, resuspended in PBS, and stained with 50 μg/ml PI in the presence of 100 μg/ml RNase A. Cell cycle distribution was analyzed using a FACSCanto flow cytometer (Becton Dickinson, San Jose, CA, USA). Data analysis was performed with the FACS Diva software (Becton Dickinson).

Caspase-3/7 activity.
To quantitate the proteolytic activity of caspase 3 and 7, we performed an assay based on the proteolytic cleavage of the C-terminal side of the aspartate residue in the DEVD peptide substrate by caspase 3/7 and into the fluorescent rhodamine 110. Caspase 3/7 activity was determined using the APO-One Homogenous Caspase-3/7 Assay (Promega Corporation, Wisconsin, USA) following the manufacturer's instructions. The obtained data were normalized to cellular protein content. The total protein was quantified by BCA assay (Pierce® BCA Protein Assay Kit, Thermo Scientific Inc., USA) according to the manufacturer's protocol.
RNA extraction and real-time PCR. Gene expression analysis was carried out according to the previously described protocol 58 . TRI reagent was applied for total RNA isolation, synthesis cDNA Transcriptor First-Strand kit (Roche Diagnostics GmbH, Mannheim, Germany) for first-strand cDNA synthesis, and SYBR® Select Master Mix (Life Technologies, Carlsbad, CA, USA) for real-time PCR. The primers used for the amplification of cDNAs are listed in Table 2. The levels of transcripts were normalized using β-actin as an internal control, which was previously applied in 12Z cells 59 . Relative mRNA expression was shown as fold change calculated using the 2 −ΔΔCt method compared with control cells.
Statistical analysis. All data are presented as means ± SD from three independent replications. Statistical analysis was performed using STATISTICA version 13.3 software (Statsoft, Inc., Tulsa, OK, USA). The Shapiro-Wilk test was used to assess distributional assumptions, and the equality of variances hypothesis was verified with Levene's test. A Student's t-test was used to compare two groups of data. One-way analysis of variance (ANOVA) followed by Tukey's post hoc test was performed to determine the differences between the mean values of multiple groups. p ≤ 0.05 was the cut-off point for a significant difference.

Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.